1. Field of the Invention
The present invention relates to a method and apparatus for distinguishing and identifying multiple subpopulations of particles in a sample without interference from other particles in the sample, and more particularly, relates to a method and apparatus for distinguishing and quantifying multiple subpopulations of cells of interest in a cell sample without interference from other cells having properties which would interfere with the quantifying of the cells of interest.
2. Description of the Prior Art
Presently known and available flow-through cytometers useful for detecting particles, cells and the like, commonly include two channels for the detection of one or more, usually two, subpopulations of cells in a mixture. For example, devices are known which include two fluorescence channels which can detect cells specifically labeled with two immunofluorescent agents associated with the respective fluorescence channels. In these known devices, a complete fluorescence channel including the electrical circuitry and fluorescence detector is used for each category of fluorochrome-treated cells to be detected in the mixture of cells in the sample being analyzed. Therefore, to detect multiple subpopulations of cells in a sample using flow-through cytometry, an equivalent number of fluorescence channels is used. A separate light source, such as a laser, is used to excite each different type of fluorochrome or immunofluorescent stain which has been tagged.
Analysis and quantifying of two different immunofluorescent stains in a single sample, utilizes two lasers providing excitation energy at significantly separated wave lengths or two fluorochromes which are activated by a single light source but which have different emission characteristics. Apparatus utilizing two lasers for analyzing an equivalent number of immunofluorescent stains are described in U.S. Pat. No. 3,826,364 and 4,284,412.
In the field of hematology in general, and in the specific field of immuno-hematology, it is desirable to determine the count of a variety of cells which circulate in the peripheral blood. Subclassification of cells is performed, and the count of the subclasses is of great interest in the evaluation of immune related disease, such as the recently recognized acquired immune deficiency syndrome (AIDS). In particular, the subclasses of the lymphocyte, a mononuclear type of white blood cell in blood, has become of great clinical significance.
There are many instances when it is desirable to be able to directly detect distinct multiple subpopulations of cells of interest from a sample, but where the sample has other cells having similar characteristics to the cells of interest interfere with such direct detection. For instance, in performing certain tests on blood, it may be desirable to detect or quantify the total population of lymphocytes cells in the blood sample and to determine the proportion of T-cells and B-cells as a percentage of the lymphocyte population. Lymphocyte cells, however, are mononuclear cells and other mononuclear cells, particularly monocytes, are present in the blood sample. Such other mononuclear cells interfere with a direct determination of T-cells and B-cells. Similarly, the detection and quantification of other different types of lymphocytes, such as the helper cell/suppressor cell subset of T-cells and the suppressor cell/natural killer cell subset of T-cells, may be desired. Monocytes would also interfere with such subset analysis. With this in mind, the present invention is directed to solving the aforementioned problems while satisfying the desired need for the direct determination of subpopulations of cells of interest from a sample mixture without interference from other cells having similar characteristics.
It would be desirable to provide an independent count of lymphocytes and of monocytes. Prior effects to provide such independent counts have concentrated on the fact that monocytes as a group are generally larger than lymphocytes. However, there is a large overlap of sizes between the lymphocytes and monocytes and such size difference methods have not been successful. In some diseases, small monocytes are abundantly present in peripheral blood and the size concept becomes completely invalid. Another means of identifying monocytes has depended on determination of an enzymatic reaction limited to monocytes. However, this means for classification and identification of monocytes is not compatible with the recognition of lymphocyte subsets.
Another aspect of the present invention is an apparatus for identifying multiple subpopulations of particles of interest in a sample containing other particles which would interfere with such identification. These particles have been selectively labeled with different marking agents having distinguishable, quantifiable characteristics capable of being stimulated. This apparatus comprises means for moving the labeled particles, substantially one at a time, in a flow path. Means is provided for stimulating the marking agents on the particles moving in the flow path. Such stimulating means is capable of stimulating the characteristics of two or more different marking agents on labeled particles. There are means for detecting the characteristics exhibited by the stimulated marking agents, means for distinguishing subpopulations of the particles relative to the detected, different quantifiable characteristics of the marking agents and means for retaining an analysis based on such detection and distinguishing means and applying the retained analysis to a subsequent or prior analysis.